Jeffrey Hoorfar

Practical considerations in design of internal amplification controls for diagnostic PCR assays.

The explosive increase since the beginning of 1990s in the number of publications reporting PCR-based methods for detection or molecular typing of food-borne pathogens has attracted the attention of end-user laboratories.nnHowever, the well recognized difficulties in reproducing published tests due

Standardization of diagnostic PCR for the detection of foodborne pathogens.

In vitro amplification of nucleic acids using the polymerase chain reaction (PCR) has become, since its discovery in the 1980s, a powerful diagnostic tool for the analysis of microbial infections as well as for the analysis of microorganisms in food samples. However, despite its potential, PCR has neither gained wide acceptance in routine diagnostics nor been widely incorporated in standardized methods. Lack of validation and standard protocols, as well as variable quality of reagents and equipment, influence the efficient dissemination of PCR methodology from expert research laboratories to end-user laboratories. Moreover, the food industry understandably requires and expects officially approved standards. Recognizing this, in 1999, the European Commission approved the research project, FOOD-PCR (http://www.PCR.dk), which aims to validate and standardize the use of diagnostic PCR for the detection of pathogenic bacteria in foods. The present review focuses on the harmonization procedure and standardization criteria for detection of foodborne pathogens by PCR. The progress of standardization so far and future perspectives of diagnostic PCR are discussed.

Diagnostic PCR: Making internal amplification control mandatory

The explosive increase since the beginning of the 1990s in the number of publications reporting PCR-based methods for detection or molecular typing of foodborne pathogens has attracted the attention of end-user laboratories. However, the well-recognized difficulties in reproducing published tests because of variation in performance of PCR thermal cyclers (Schoder et al. 2003), in efficiency of different DNA polymerases, and in the presence of PCR inhibitors in the sample matrix, have hampered implementation in end-user laboratories. This particularly applies to laboratories with quality-assurance programmes. It is necessary to have PCR-based methods available as internationally recognized standards (Hoorfar and Cook 2002). Currently, lack of international standards often forces end-user laboratories to spend substantial resources on adaptation of the published tests. Although many commercial PCR kits are available, it is important that end-users and reference laboratories have access to open-formula, noncommercial and nonproprietary PCRs in which the information on target gene and reagents are fully available. The prerequisite for a PCR, published in the scientific literature, to be adopted as a standard is that it has to be nonproprietary, and to have been validated through multicentre collaborative trial according to the international criteria (Anon. 2001, 2002a; Hoorfar and Cook 2002). Multicentre trial validation of noncommercial PCRs for detection of zoonotic pathogens has been performed by a European validation and standardization project (FOODPCR: http://www.pcr.dk) involving 35 laboratories from 21 countries (Hoorfar 1999; Malorny et al. 2003). A major drawback of most published PCRs, surprisingly even to date, is that they do not contain an internal amplification control (IAC). An IAC is a nontarget DNA sequence present in the same sample reaction tube, which is co-amplified simultaneously with the target sequence. In a PCR without an IAC, a negative response (no band or signal) can mean that there was no target sequence present in the reaction. But, it could also mean that the reaction was inhibited, as a result of malfunction of thermal cycler, incorrect PCR mixture, poor polymerase activity and, not least, the presence of inhibitory substances in the sample matrix. Conversely, in a PCR with an IAC, a control signal will always be produced when there is no target sequence present. When neither IAC signal nor target signal is produced, the PCR reaction fails. Thus, when using a PCRbased method in routine analysis, an IAC, if the concentration adjusted correctly, will indicate false-negative results. It is the false-negative results that turn a risk into a threat for the population, whereas a false-positive result merely leads to a clarification of the presumptive results by re-testing the sample. The European Standardization Committee (CEN), in collaboration with International Standard Organization (ISO) has proposed a general guideline for PCR testing that requires the presence of IAC in the reaction mixture (Anon. 2002b). Therefore, only IAC-containing PCRs may undergo multicentre collaborative trials, which is a prerequisite for standardization. Scientific journals must provide the source of new PCRbased methods suitable for standardization. Therefore, we propose that henceforward the editorial boards of applied microbiology journals require inclusion of an IAC in diagnostic PCR reported in submitted manuscripts. This could be performed by providing a specific section devoted to PCR in their Instruction to Authors.

Toward an international standard for PCR-based detection of food-borne thermotolerant Campylobacters: assay development and analytical validation.

ABSTRACT As part of a European research project (FOOD-PCR), we developed a standardized and robust PCR detection assay specific for the three most frequently reported food-borne pathogenic Campylobacter species, C. jejuni, C. coli, and C. lari. Fifteen published and unpublished PCR primers targeting the 16S rRNA gene were tested in all possible pairwise combinations, as well as two published primers targeting the 23S rRNA gene. A panel of 150 strains including target and nontarget strains was used in an in-house validation. Only one primer pair, OT1559 plus 18-1, was found to be selective. The inclusivity and exclusivity were 100 and 97%, respectively. In an attempt to find a thermostable DNA polymerase more resistant than Taq to PCR inhibitors present in chicken samples, three DNA polymerases were evaluated. The DNA polymerase Tth was not inhibited at a concentration of 2% (vol/vol) chicken carcass rinse, unlike both Taq DNA polymerase and DyNAzyme. Based on these results, Tth was selected as the most suitable enzyme for the assay. The standardized PCR test described shows potential for use in large-scale screening programs for food-borne Campylobacter species under the assay conditions specified.

Diagnostic PCR: comparative sensitivity of four probe chemistries.

Three probe chemistries: locked nucleic acid (LNA), minor groove binder (MGB) and Scorpion were compared with a TaqMan probe in a validated real-time PCR assay for detection of food-borne thermotolerant Campylobacter. The LNA probe produced significantly lower Ct-values and a higher proportion of positive PCR responses analyzing less than 150 DNA copies than the TaqMan probe. Choice of probe chemistry clearly has an impact on the sensitivity of PCR assays, and should be considered in an optimization strategy.

Physical Characteristics of Six New Thermocyclers

Since the publication of the first article describing PCR, thermocyclers have become a staple in academic and industrial laboratories (1). The thermocycler is a programmable cycling incubator that performs repeated PCR steps of DNA denaturation, primer annealing, and primer elongation at defined intervals. Rapid heat transfer from the heating block to the in-tube sample liquid ensures a high efficiency of amplicon multiplication; therefore, a thermal processor should guarantee temperature uniformity for all samples within an individual run as well as run-to-run repeatability.nnPCR-based protocols can give unsatisfying results (2)(3). Several collaborative studies have shown weak reproducibility with random amplified polymorphic DNA (RAPD) protocols (4)(5). One reason might be the influence of the thermocycler on amplification efficiency.nnDespite its striking importance for PCR, the literature on thermocyclers is scarce. Some studies were published on the first generation of cyclers (6)(7)(8). Others determined the amplification efficiency but did not evaluate the physical characteristics (9)(10). The impact on PCR of the variation within thermocyclers, with regard to their thermocycling settings, has not been fully determined. The goal of this study was to define the physical characteristics of performance of the latest generation of thermocyclers and to discuss the influence of the physical properties on amplification efficiency.nnSix new thermocyclers were selected for this performance study: ( A ) Gene Amp 9700 (Applied Biosystems), ( B ) Multicycler PTC 200 (MJ Research, Inc.), …

Diagnostic PCR: making internal amplification control mandatory.

The explosive increase since the beginning of the 1990s in the number of publications reporting PCR-based methods for detection or molecular typing of foodborne pathogens has attracted the attention of end-user laboratories. However, the well-recognized difficulties in reproducing published tests because of variation in performance of PCR thermal cyclers (Schoder et al. 2003), in efficiency of different DNA polymerases, and in the presence of PCR inhibitors in the sample matrix, have hampered implementation in end-user laboratories. This particularly applies to laboratories with quality-assurance programmes. It is necessary to have PCR-based methods available as internationally recognized standards (Hoorfar and Cook 2002). Currently, lack of international standards often forces end-user laboratories to spend substantial resources on adaptation of the published tests. Although many commercial PCR kits are available, it is important that endusers and reference laboratories have access to openformula, noncommercial and nonproprietary PCRs in which the information on target gene and reagents are fully available. The prerequisite for a PCR, published in the scientific literature, to be adopted as a standard is that it has to be nonproprietary, and to have been validated through multicentre collaborative trial according to the international criteria (Anon. 2001, 2002b; Hoorfar and Cook 2002). Multicentre trial validation of noncommercial PCRs for detection of zoonotic pathogens has been performed by a European validation and standardization project (FOOD-PCR: http://www.pcr.dk) involving 35 laboratories from 21 countries (Hoorfar 1999; Malorny et al. 2003). A major drawback of most published PCRs, surprisingly even to date, is that they do not contain an internal amplification control (IAC). An IAC is a nontarget DNA sequence present in the same sample reaction tube, which is co-amplified simultaneously with the target sequence. In a PCR without an IAC, a negative response (no band or signal) can mean that there was no target sequence present in the reaction. But, it could also mean that the reaction was inhibited, as a result of malfunction of thermal cycler, incorrect PCR mixture, poor polymerase activity and, not least, the presence of inhibitory substances in the sample matrix. Conversely, in a PCR with an IAC, a control signal will always be produced when there is no target sequence present. When neither IAC signal nor target signal is produced, the PCR reaction fails. Thus, when using a PCR-based method in routine analysis, an IAC, if the concentration adjusted correctly, will indicate false-negative results. It is the false-negative results that turn a risk into a threat for the population, whereas a false-positive result merely leads to a clarification of the presumptive results by re-testing the sample. The European Standardization Committee (CEN), in collaboration with International Standard Organization (ISO) has proposed a general guideline for PCR testing that requires the presence of IAC in the reaction mixture (Anon. 2002a). Therefore, only IAC-containing PCRs may undergo multicentre collaborative trials, which is a prerequisite for standardization. Scientific journals must provide the source of new PCRbased methods suitable for standardization. Therefore, we propose that henceforward the editorial boards of applied microbiology journals require inclusion of an IAC in diagnostic PCR reported in submitted manuscripts. This could be performed by providing a specific section devoted to PCR in their Instruction to Authors.

Novel Approach for Assessing Performance of PCR Cyclers Used for Diagnostic Testing

ABSTRACT As part of a large international project for validation and standardization of PCR, the influence of thermocyclers on PCR was tested. Six brand-new, Peltier technology-driven 96-well thermocyclers were subjected to a novel and stringent in-tube (not block) physical testing. The temperature was directly monitored in PCR tubes containing 50 μl of distilled water at 13 different block positions. The certified temperature accuracy of the measurement system was ±0.3°C. Finally, the results of the physical testing were compared to those of an amplification efficiency study running an in-house PCR assay. The cyclers did not perform within the manufacturer′s specification. Premature timing, under- and overshooting, and spatial variation of heat transfer were found to be the critical factors. The physical testing allowed us to distinguish accurate from less-accurate (2/6) cyclers. The lack of thermal homogeneities became most evident at the denaturation level during the first 15 s. At the time point zero, the accurate cyclers showed temperature deviations of 0.5 to 1.5°C, whereas less-accurate cyclers failed to reach the set temperature by 13 to 20°C. Consequently, the two less-accurate cyclers could not gain positive PCR results by running an in-house PCR assay. However, by modifying the original temperature protocol by increasing the denaturation temperature and time, the amplification efficiency of these two cyclers could be improved significantly. The results have implication for laboratories using diagnostic PCR testing.